Materials and Reagents

M-CSF-transduced L929 cells
HI FBS (EuroClone, catalog number: EC S0180L )
L-Glutamine (EuroClone, catalog number: EC B3000D )
Penicillin/streptomycin (EuroClone, catalog number: EC B3001D )
IMDM (EuroClone, catalog number: EC B2072L )
Beta-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
M-CSF-transduced L929 growth supernatant
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECM9605AX )
BMMFs culture medium/ conditioned medium (see Recipes)

Equipment

Centrifuges
70 μm-wide cut-off cell strainer
Non-treated cell culture plates
Incubator (37 °C and 5% CO₂)
Fluorescence activated cell sortor (FACS)

Procedure

Flush mouse tibiae and femurs with ice-cold PBS through a 70 μm-wide cut-off cell strainer.
Centrifuge 5 min at 450 x g. Resuspend pelleted cells in conditioned medium (supplemented with 30% of growth supernatant of M-CSF-transduced L929 cells).
Seed 7 x 10⁶ cells in 100 x 20 mm non-treated cell culture plates in 10 ml of conditioned medium.
Incubate at 37 °C and 5% CO₂.
Upon reaching confluence (approximately 6 days) use the cells or split adhered cells and seed 5 x 10⁶ cells in 100 x 20 mm in non-treated cell culture plates in 10 ml of conditioned medium.
BMMFs are ready for experimental use when the percentage of CD11b⁺ cells is higher than 90% as measured by FACS analysis.

Recipes

BMMFs culture medium recipe (conditioned medium)
HI FBS - 10%
L-Gln - 2mM
Penicillin/streptomycin - 50 U/ml
Beta-mercaptoethanol - 50 μM
M-CSF-transduced L929 growth supernatant - 30%
IMDM - to volume

Acknowledgments

This protocol is described in our Nature paper (Zanoni et al., 2009). This work was supported by grants from the CARIPLO Foundation, the European Commission 6th Framework Program (MUGEN and DC-THERA contracts), the European Commission 7th Framework Program (TOLERAGE and ENCITE contracts), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the and the Italian Ministry of Education and Research (COFIN).

References

Zanoni, I., Ostuni, R., Capuano, G., Collini, M., Caccia, M., Ronchi, A. E., Rocchetti, M., Mingozzi, F., Foti, M., Chirico, G., Costa, B., Zaza, A., Ricciardi-Castagnoli, P. and Granucci, F. (2009). CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Nature 460(7252): 264-268.